Peter Ruef

Endotoxin binding to erythrocyte membrane and erythrocyte deformability in human sepsis and in vitro

ObjectiveSeveral studies have shown that lipopolysaccharide and lipid A impair red blood cell deformability in vitro and in vivo. However, it is unclear whether impaired red blood cell deformability is associated with binding of lipopolysaccharide to the red blood cell membrane. DesignAnalysis of hydroxymyristic acid content in red blood cell membranes and red blood cell deformation in patients with Gram-negative septicemia and after in vitro incubation of red blood cells from healthy adults with 100 &mgr;g of Escherichia coli lipid A or 1 mg of E. coli lipopolysaccharide per milliliter of red blood cell in buffer solution and in whole blood. Hydroxymyristic acid is a fatty acid of the lipid A part of lipopolysaccharide in most Gram-negative bacteria. SettingUniversity research laboratories. SubjectsTen healthy adults and four patients with clinical and laboratory signs of septicemia. InterventionsBlood sampling. Measurements and Main ResultsRed blood cell deformation was measured with a laser-diffraction shearing device (Rheodyn) and a computerized micropore filtration system (CTA). Lipopolysaccharide and lipid A binding to red blood cell membranes was studied by measuring the amide-linked hydroxymyristic acid by gas chromatography.The detection rates of hydroxymyristic acid were 82% for lipopolysaccharide and 79% for lipid A in buffer solution. In membranes of washed red blood cell, the detection rates of lipopolysaccharide and lipid A were 0.26 ± 0.03% (2.6 ± 0.3 &mgr;g/mL) and 1.3 ± 0.5% (1.3 ± 0.5 &mgr;g/mL), and in red blood cell membranes of whole blood the detection rates were 2.6% (25.5 &mgr;g/mL) and 4.1% (4.1 &mgr;g/mL), respectively. The lipopolysaccharide content in red blood cell membranes of septic patients ranged from 47 to 103 &mgr;g/mL of red blood cell. Red blood cell deformation in the Rheodyn and in the CTA were not influenced by lipopolysaccharide incubated with washed red blood cells. In the Rheodyn, red blood cell deformation was significantly decreased by 18% after lipid A incubation in washed red blood cells, by 26% after lipopolysaccharide incubation in whole blood, and by 31% in septic patients. Similar effects were observed when we used the CTA. ConclusionsRed blood cell deformation is decreased in septic patients, after in vitro incubation of washed red blood cells with lipid A and of whole blood with lipopolysaccharide. Lipopolysaccharide did not influence red blood cell deformation after incubation with washed red blood cells. The decrease of red blood cell deformation was related to the amount of hydroxymyristic acid measured in red blood cell membranes, suggesting that endotoxin binding directly affects mechanical properties of red blood cells.

Passive deformability of mature, immature, and active neutrophils in healthy and septicemic neonates

Obstruction of narrow vessels by rigid neutrophils may contribute to ischemic organ injury. In septicemia, a substantial portion of the neutrophils may become activated and the number of circulating immature neutrophils may rise sharply. Volume and deformability of mature (PMN) and immature neutrophils in healthy preterm and full-term infants and in septicemic neonates were studied by means of a micropipette system. Membrane cytoplasm tongues were aspirated into 2.5-µm (diameter) pipettes over a period of 60 s. Volume and tongue growth of mature resting PMN were similar in healthy preterm and full-term neonates and adults. Compared with mature PMN (about 360 fl), the volumes of band cells (415 fl), metamyelocytes (470 fl), and less mature cells (myeloblasts, promyelocytes, and myelocytes; 490 fl) were significantly increased (p < 0.005). Final tongue lengths of band cells, metamyelocytes, and less mature cells were decreased by about 50, 60, and 70%, respectively, when compared with passive mature cells. In septic neonates, the percentage of immature neutrophils was increased, but the deformability and volume of the cell subpopulations were not affected by septicemia. Active PMN were characterized by pseudopod formation. More active PMN were found in group B streptococcal (14% of total PMN), Gram-negative (12%), and Staphylococcus epidermidis septicemia (8%) than in healthy neonates and adults (4%). The main bodies of active PMN were less deformable than passive PMN, and the pseudopods showed very little membrane deformation. The increased number of rigid active and immature neutrophils may contribute to impaired microcirculation and the high risk for organ injury in septic patients.

Impaired deformability of erythrocytes and neutrophils in children with newly diagnosed insulin-dependent diabetes mellitus

Aims/hypothesis. Abnormal rheological properties of erythrocytes, leucocytes and plasma may have a role in the development of diabetic microangiopathy. We hypothesized that changed haemorrheological variables may already be found in children with onset diabetes. Methods. Erythrocyte deformation (rheoscope), neutrophil deformation (micropipette), erythrocyte aggregation, blood and plasma viscosity were measured in 15 children with insulin-dependent diabetes mellitus before initiation of insulin treatment and 4 to 6 weeks later, 15 diabetic children treated with insulin for 5 to 8 years, 15 healthy children and 15 healthy adults. Results. At a low shear stress of 0.6 Pa, erythrocyte deformation was decreased in the diabetic children before (–28 %), after 4 to 6 weeks (–22 %) and after 5 to 8 years (–17 %) of insulin treatment compared with healthy children. More active neutrophils were counted in the untreated diabetic children (9 ± 6 %) than in healthy children (3 ± 2 %). Deformability of passive neutrophils was greatly decreased in the children with onset diabetes and moderately reduced in the diabetic children who were treated with insulin. Neutrophil deformation (r = –0.52) and erythrocyte deformation at 0.6 Pa (r = –0.62) were inversely related to haemoglobin A1 c. Haematocrit and blood viscosity were increased in the untreated children and in the children treated with insulin for 5 to 8 years. Plasma viscosity and erythrocyte aggregation were similar in the three groups of children. Conclusion/interpretation. Decreased erythrocyte deformation at low shear force, increased count of active neutrophils and impaired deformability of passive neutrophils may increase the risk for acute cerebro-vascular complications in children with uncontrolled insulin-dependent diabetes mellitus. [Diabetologia (1999) 42: 865–869]

Deformability and geometry of neonatal erythrocytes with irregular shapes

More stomatocytes, spherocytes, and erythrocytes with spicules and protrusions are found in blood from newborn infants than in blood from adults. The objective of this study was to measure volume, surface area, membrane rigidity, and viscosity of red blood cells (RBC) with irregular shapes in preterm and full-term neonates. RBC from 13 full-term and 12 preterm (25-36 wk of gestation) infants were studied by means of a micropipette system. In full-term neonates and in preterm infants, 19 and 26% of RBC, respectively, showed shapes different from those of discocytes. Keratocytes, spherocytes, akanthocytes (in full-term infants), and elliptocytes (in preterm infants) had smaller volumes than did discocytes (p < 0.05). Spherocytes showed decreased and elliptocytes had increased excess surface area and swelling capacity compared with discocytes, indicating both volume and membrane loss for spherocytes and only volume loss for elliptocytes. Immature RBC and knizocytes had similar large volumes and surface areas, suggesting that knizocytes in neonates are relatively young RBC. Increased membrane rigidity was found only in stomatocytes. Membrane viscosity was decreased in immature RBC and knizocytes and increased in stomatocytes, keratocytes, akanthocytes, and elliptocytes when compared with discocytes. There were inverse relationships between RBC volume and membrane rigidity (r = -0.42) and between RBC volume and membrane viscosity (r = -0.72). This suggests that volume loss of neonatal RBC is associated with impaired membrane deformability. Moreover, we conclude that the geometry and membrane deformability of some of the irregularly shaped neonatal RBC resemble properties of RBC in hemolytic anemias (e.g. spherocytosis).

Deformability and volume of neonatal and adult leukocytes.

ABSTRACT: Volume and deformability of blood cells are important determinants of the microcirculation. Leukocytes are larger and considerably less deformable than erythrocytes. In our study, volume and deformability of polymorphonuclear neutrophils (PMN), lymphocytes, and monocytes in adults and full-term neonates were studied by means of a micropipette system. Neonatal immature granulocytes were also investigated. Membrane cytoplasm tongues were aspirated into 2.5-μm (diameter) micropipettes over a period of 1 min. Adult and neonatal PMN were totally aspirated into 5-μm micropipettes. Tongue growth and final tongue length of PMN were about twice those of monocytes and lymphocytes. At a pressure of −2 cm H2O, tongue growth of lymphocytes and monocytes was similar. At a pressure of −4 cm 2O, however, tongue growth of monocytes was faster and the final tongue was longer than those of lymphocytes (p < 0.05). Cellular volume and deformation behavior of the different leukocyte subpopulations (PMN, monocytes, and lymphocytes) were similar in neonates and adults. Compared to mature neonatal PMN, immature neonatal neutrophilic granulocytes were significantly less deformable (final tongue length of 5.4 ± 1.52 versus 9.3 ± 1.48 pm at −2 cm H2O) and larger (421 ± 68 versus 360 ± 38 fL). The entry time of PMN into 5-pm micropipettes was similar in neonates and adults at aspiration pressures of −2, −3, and −4 cm H2O. We conclude that the deformability of neonatal and adult leukocytes is not different despite functional differences and that immature granulocytes may contribute to impaired microcirculation in neonates with severe septicemia or hypoxemia.

Dietary docosahexaenoic acid improves red blood cell deformability in rats

Abstract Epidemiologic studies have shown that consumption of fish rich in the two n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), decreases the mortality from cardiovascular disease (1,2). The n-3 fatty acids alter the formation of eicosanoids, modulators involved in regulation of blood vessel diameter and inflammation. By ingestion of n-3 fatty acids, the eicosanoid system is shifted toward vasodilation, antiaggregation of platelets, and less proinflammation by leukotrienes B (3). The mechanisms involved are many-fold varying from actions on platelets, plasma lipid pattern and endothelial function. Red blood cell (RBC) deformability may also be improved by fish oil as suggested by filtration studies (4,5,6). However, fish oil contains both n-3 fatty acids, i.e. EPA and DHA. Studies on the effect of individual omega-3 fatty acids on RBC deformability are lacking. This study was designed to investigate the effect of diets supplemented with purified n-3 fatty acids (EPA and DHA) alone or in combination on RBC membrane n-3 fatty acids and RBC deformation in rats.

LPS- and LTA-Induced Expression of IL-6 and TNF-α in Neonatal and Adult Blood: Role of MAPKs and NF-κB

As nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) seem to be critical mediators in the inflammatory response, we studied the effects of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) on (a) the activation of NF-κB and MAPKs and (b) the expression of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) with or without the specific inhibitors of these intracellular signal transduction pathways in neonatal cord and adult blood. TNF-α and IL-6 concentrations showed a sharp increase in the supernatants of cord and adult whole blood after stimulation. TNF-α concentrations were significantly higher, whereas IL-6 concentrations were tendentially lower in adult blood after stimulation. Stimulation with LPS or LTA resulted in a significantly decreased activation of p38 MAPK in neonatal compared with adult blood. Although LTA failed to induce additional ERK1/2 phosphorylation, LPS stimulation mediated the moderately increased levels of activated ERK1/2 in neonatal monocytes. The addition of the p38 MAPK inhibitor SB202190 significantly decreased IL-6 and TNF-α production upon LPS or LTA stimulation. Furthermore, the inhibition of ERK1/2 was able to reduce LPS-stimulated TNF-α production in neonatal blood. We conclude that p38 MAPK as well as ERK1/2 phosphorylation is crucially involved in LPS activation and could explain the differences in early cytokine response between neonatal and adult blood.

CXCL1-Triggered Interaction of LFA1 and ICAM1 Control Glucose-Induced Leukocyte Recruitment during Inflammation In Vivo

It is well acknowledged that proinflammatory stimulation during acute hyperglycemia is able to aggravate inflammatory diseases. However, the mechanisms of proinflammatory effects of glucose are controversially discussed. We investigated leukocyte recruitment after intravenous injection of glucose in different inflammatory models using intravital microscopy. Flow chamber experiments, expression analysis, functional depletion, and knockout of key adhesion molecules gave mechanistic insight in involved pathways. We demonstrated that a single injection of glucose rapidly increased blood glucose levels in a dose-dependent manner. Notably, during tumor necrosis factor (TNF) α-induced inflammation leukocyte recruitment was not further enhanced by glucose administration, whereas glucose injection profoundly augmented leukocyte adhesion and transmigration into inflamed tissue in the trauma model, indicating that proinflammatory properties of glucose are stimulus dependent. Experiments with functional or genetic inhibition of the chemokine receptor CXCR2, intercellular adhesion molecule 1 (ICAM1), and lymphocyte function antigen 1 (LFA1) suggest that keratino-derived-chemokine CXCL1-triggered interactions of ICAM1 and LFA1 are crucially involved in the trauma model of inflammation. The lacking effect of glucose on β 2 integrin expression and on leukocyte adhesion in dynamic flow chamber experiments argues against leukocyte-driven underlying mechanisms and favours an endothelial pathway since endothelial ICAM1 expression was significantly upregulated in response to glucose.

Effect of activators and the phosphodiesterase inhibitors pentoxifylline and enoximone on the deformability of neutrophils in neonates and adults.

Background: Stimulated polymorphonuclear leukocytes (PMN) are extremely rigid compared to resting PMN. They may obstruct narrow vessels and contribute to ischaemic organ injury. Deformability is a prerequisite for both active and passive movement in the microcirculation. Aim: The investigation was designed to study whether stimulators and inhibitors of stimulation show different effects on deformability of neonatal and adult PMN. Methods: Deformability of PMN was assessed by complete aspiration of a PMN into a micropipette with an internal diameter of 5 μm. Blood samples from 20 neonates and 20 adults were studied before and after stimulation with N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), interleukin‐8 (IL‐8) or tumour necrosis factor‐α (TNF‐α). Moreover, effects of the phosphodiesterase inhibitors Pentoxifylline (PTX) and Enoximone on the deformability of stimulated PMN were investigated. Results: fMLP, IL‐8 and TNF‐α significantly delayed aspiration times of PMN in relation to the concentrations of the stimulators. The addition of PTX or Enoximone to stimulated PMN increased the deformability up to 60% depending on the concentration of the inhibitors. No significant differences in the aspiration times were found between neonatal and adult PMN at any of the experimental conditions after activation with the three stimulators and treatment with the two inhibitors.

Anti-inflammatory effects of selected drugs on activated neonatal and adult neutrophils

Abstract Aim. In view of the central role of granulocytic neutrophils in the context of inflammatory reactions, the present study focuses on anti-inflammatory effects of drugs on activated neutrophils in neonates and adults. Methods. Sixteen blood samples of neonates and adults were investigated in a prospective study. Loss of deformability, morphological changes, and increases in neutrophil elastase were determined as measures of neutrophil activation due to incubation with the pro-inflammatory cytokine interleukin-8. For inhibition experiments, the blood samples were also incubated with the phosphodiesterase inhibitors milrinone and piclamilast, the protease inhibitor urinastatin, ketamine, protein C concentrate, and the nitric oxide donor FK 409. Changes in deformability were investigated with a cell transit analyzer, morphological changes by microscopic observation, and the extent of neutrophil elastase release with an enzyme immunoassay. Results. The drugs milrinone, piclamilast, urinastatin, ketamine, protein C concentrate and FK 409 showed deactivating effects on activated neutrophils in recommended clinical doses. They improved deformability as well as reduced pseudopod formation and the release of neutrophil elastase. The effects on neutrophils did not differ between neonates and adults despite their functional differences. Conclusion. We conclude that these drugs may reduce the inflammatory response and improve microcirculation in neonates and adults during inflammation.